Non-small cell lung cancer (NSCLC), the most common form of lung cancer, is treated with tumor resection and chemotherapy, with ionizing radiation (IR) treatment and chemotherapy used for patients with unresectable tumors. Despite improvements in medical management, 5-year survival rates for NSCLC remain low. Evidence suggests that resistance to radio- and chemotherapy is associated with cancer stem cells (CSCs), which are undifferentiated cells with the capacity to self-renew and differentiate, restoring the tumor cell population following treatment. However, the mechanisms through which lung CSCs evade and resist radiation therapy remain completely uncharacterized. We previously reported on our ability to isolate, culture, and assay human lung CSCs from cultured cell lines. We found stem cell factor (SCF) and its receptor c-kit function in a crucial pathway for the self-renewal and proliferation of human lung CSCs. We discovered that a combination of cisplatin (to eradicate bulk tumor cells) and agents targeting SCF/c-kit signaling in CSCs (e.g., antibody neutralizing SCF and anti-c-kit RTK inhibitors) is an effective therapy against NSCLC in vitro. In the proposed study, we will extend our work to CSCs derived from primary NSCLC surgical tumor specimens to test following hypotheses: 1) IR treatment selects for lung CSCs; 2) CSC radioresistance is mediated by preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity; 3) SCF/c-kit signaling is a central component of self renewal and survival of CSCs in lung tumors; and 4) The combination of IR treatment and SCF/c-kit pathway inhibition will eliminate bulk NSCLC cells and CSCs. Our specific aims are to: 1. Investigate CSC resistance to IR and characterize the molecular mechanisms associated with radioresistance; and 2. Investigate SCF/c-kit signal transduction in lung CSCs and the death signaling mechanisms activated in CSCs treated with neutralizing SCF antibody or anti-c-kit RTK inhibitors. To determine whether targeting SCF/c-kit signaling with these agents will enhance the effectiveness of IR treatment of NSCLC in vitro. We will generate new knowledge regarding lung CSC biology, and our findings could lead to significant improvements in the efficacy of NSCLC radiation therapy.